The invention relates to the field of high performance liquid chromatography and more particularly to the field of high performance liquid chromatography separation of small organic molecules.
High-performance liquid chromatography (HPLC) is commonly used for analytical and preparative separations of biopolymers and other organic molecules. For instance, the individual components within a complex organic reaction mixture may be separated by HPLC. HPLC is performed in a pressure-resistant tube containing a stationary adsorbent which is the packing material. A pressure mechanism exerts pressure on a mobile phase applied to one end of the column and moves it through the column causing it to exit the opposite end of the column. A sample containing a mixture of compounds is injected onto the column through a sample injection port. As the sample moves through the packing material, the various components of the sample adsorb to the packing material with different affinities. The components, therefore, can elute from the column separately under appropriate conditions. On a reverse phase HPLC column the compounds within a sample are separated based on hydrophobicity.
HPLC analysis may be performed in isocratic or gradient mode. An isocratic HPLC separation is one which is carried out under a constant eluant composition. A gradient HPLC separation is characterized by a gradual change in the percentage of two or more solvents applied to the column over time. The change in solvent often is controlled by a mixing device which mixes solvent A and solvent B to produce the HPLC solvent just prior to its movement through the column. The amount of time over which the gradient is changed from one extreme to the opposite extreme is the gradient time.
Generally in gradient chromatography it is believed that increasing the flow rate and/or decreasing the gradient time results in a loss of resolution, that is the ability of the column to separate the components within the mixture into discrete eluant fractions. (Snyder, L., et al., xe2x80x9cPractical HPLC Method Developmentxe2x80x9d, Wiley-Interscience Publication, (1997).
Rapid methods for the preparation and isolation of potential drug candidates using automated synthetic organic chemistry techniques to create combinatorial libraries represents an important advance in drug discovery. Certain combinatorial libraries encompass a series of compounds having common structural features but which differ in the number or type of group attached to the main structure. Each compound within a combinatorial library created by parallel synthesis is a separate sample housed in a tube or well of a microtitre plate. Once the library is completed, each sample is subjected to quality control analysis to confirm that the particular sample includes the desired library component at the requisite purity. Generally this is accomplished by subjecting the samples to HPLC with UV, evaporative light scatter detection (ESLD), or mass spectrometry detection; IR; NMR; or any other appropriate analytical techniques. The qualitative analysis of such combinatorial libraries by conventional HPLC requires on the average 5 to 20 minutes in order to separate various compounds within the sample.
A problem encountered with prior art methods for separation of compounds in combinatorial libraries using HPLC is the length of time required for separation of each sample. Each sample of a combinatorial library produced by parallel synthesis must be analyzed separately to determine if that sample houses the appropriate compound and/or to separate the compounds in the mixture. Each library includes thousands of samples each of which require an average run time of 10 minutes. The amount of time required to perform separations on these samples may run on the order of months using standard equipment and methodology.
The present invention provides rapid methods for the analysis and preparative isolation of relatively simple synthetic mixtures containing a small number of reagents, the product(s) of interest and a relatively small number of side products using HPLC. The methods of the invention reduce the HPLC analysis run time per sample from an average of 5-20 minutes shown in the prior art (Weller, et al., Molecular Diversity, (1997), 3:61-70) to less than one minute without a meaningful loss of resolution. The invention depends in part upon the discovery that small organic molecules could be separated on a full gradient reverse phase HPLC by minimizing the total volume of eluant applied to the column, maximizing the linear flow velocity of the eluant and compressing the gradient time to resolve a peak at least every 2 seconds. A full gradient is defined as a change in the solvent B concentration of at least 50%. For example, if the initial concentration of solvent B was 15%, a full gradient would be achieved when the concentration of solvent B reached 65%. The prior art believed that if the total eluant volume was decreased and the flow rate increased to the levels indicated in the invention, the resolution of the peaks eluting off the column would be significantly decreased to an extent that it would not be possible to obtain a discrete separation of a mixture of small organic compounds.
The methods of the invention include applying a mixture of compounds to a reverse phase column configured in a gradient high performance liquid chromatography system, and operating with a flow rate of at least 5 column volumes/min. A complete gradient is applied to the column at a rate which uses a maximum total volume of 10 column volumes; preferably 5 column volumes in order to maximize speed. These parameters allow each small organic component within the mixture of compounds to elute in a distinct fraction from the column with sufficient resolution which permits a peak production of at least 1 peak/2 seconds. A one minute analysis, using a peak production of 1 peak/2 seconds, would translate into an analysis which could baseline separate more than 30 individual peaks.
The amount of time that the complete separation requires depends on the parameters used in the separation, such as the length of the column and the amount of solvent used. Preferably the mixture of compounds is applied to the column at a first time point and all the compounds are eluted within a time period of less than one minute from the first time point. In other preferred embodiments all compounds are eluted within a time period of less than 30 seconds. In other embodiments all compounds are eluted within a time period of less than 20 seconds.
In one embodiment of the invention the method also includes the step of detecting at least one of the compounds as it elutes from the column. In another embodiment the method includes the step of collecting at least one of the compounds in a distinct fraction as it elutes from the column.
In preferred embodiments, the mixture of molecules includes reactants and a substantially pure product of the reactants.
In other preferred embodiments, including those listed above, the column is less than or equal to 30 mm in length. The column is less than or equal to 15 mm in length in other embodiments.
According to other preferred embodiments, including those listed above, the column has a packing material which has an average diameter of less than 5 microns.
In other preferred embodiments, including those listed above, the peak production is at least 1 peak/1 second. The peak production is at least 1 peak/0.5 seconds in other embodiments.
Preferably the total volume of liquid applied to the column per analysis is less than 15xc3x97 column volumes, preferably less than 8xc3x97 column volumes. The total volume of liquid may include a cleaning volume having a maximum of 2xc3x97 column volume. In a preferred embodiment the total volume of liquid may include an equilibration volume having a maximum of 1xc3x97 column volume.
In other embodiments, including those listed above, the mixture of molecules is a member of a combinatorial library of small organic molecules. Preferably the combinatorial library is made by means of parallel synthesis methods and the method is performed for high throughput purification and/or quality control analysis.
In additional embodiments the column flow rate has a linear velocity of at least 3 mm/sec. In another embodiment the linear velocity is 5 mm/sec.
The small organic compounds which elute from the column are typically analyzed by a detection device such as a UV detector. In one embodiment of the invention the small organic molecules are analyzed by both a UV detector and a mass spectrometer.
In one embodiment the method is a method for analysis of at least one compound in the mixture of compounds. Preferably less than 10 xcexcg of the mixture of compounds is applied to the column for the analysis. In an embodiment the sample of compounds is not collected for further use after it is eluted from the column.
In another embodiment the method is a method for preparative isolation of at least one compound in the mixture of compounds. Preferably between 1 and 100 mg of the mixture of compounds is applied to the column for the analysis. In an embodiment the compounds are collected in separate fractions for further use after they are eluted from the column. Preferably the at least one compound is collected in a fraction having a volume of 2 milliliters or less.
The high performance liquid chromatography may be performed at a temperature of greater than 20xc2x0 C. in one embodiment. In another embodiment the method is performed at a temperature of greater than 50xc2x0 C. In a preferred embodiment the method is performed at a temperature of greater than 60xc2x0 C.
In another aspect the invention is a rapid high performance liquid chromatography method for the preparative isolation of a concentrated fraction of a small organic compound from a mixture of compounds. The method includes the steps of applying the mixture of compounds to a reverse phase column in a gradient high performance liquid chromatography system, wherein the column has a flow rate of at least 5 column volumes/min, applying a complete gradient to the column in a maximum volume of 10xc3x97 column volume, causing the small organic compound to elute in a distinct fraction, separate from the other molecules, from the column such that the elution permits resolution with a peak production of at least 1 peak/4 seconds and collecting the small organic compound. The small organic compound, in some embodiments is collected in a fraction having a maximum volume of 2 milliliters.
In other embodiments, including those listed above, the mixture of molecules is a member of a combinatorial library of small organic molecules. Preferably the combinatorial library is made by means of parallel synthesis methods and the method is performed for high throughput purification and/or quality control analysis.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each method.